Recently, the cDNA of TrxR1 from human placenta was cloned and sequenced. An additional redox pair at the C-terminus of the enzyme was found to consist of Cys-Secys. The C-terminus extension and the C-terminus redox pair account for the wide substrate specificity and are the main features that differentiate mammalian thioredoxin reductase from the smaller prokaryotic and yeast enzymes. To further study the role of selenocysteine in electron transfer from NADPH to substrate, we overexpressed the wild type and Secys/Cys mutant of human cytosolic TrxR1 in E. coli. A selenocysteine insertion sequence element (SECIS) required for selenocysteine incorporation in E. coli formate dehydrogenase (fdh) or minimal SECIS (Z. Liu et al., 1998) was inserted downstream of the TGA codon after the gene-specific sequence. Wild type without SECIS, wild type with SECIS or minimal SECIS, and the Secys/Cys TrxR1 mutant were amplified by PCR and ligated to different plasmids to enhance solubilization and purification yield of expressed proteins. In order to increase solubilization and correct folding of the TrxR1 expressed in E. coli, recombinant protein was fused to a calmodulin binding protein or to a His-tag and variety of cultural conditions were investigated. The solubility of hTrxR1, the specific activity, and the extent of FAD incorporation increased when cells were grown at low temperature (18-25C) in rich LB media and low concentrations of IPTG used for induction. The optimized growth conditions allowed purification to apparent homogenicity of about 4-5 milligrams of recombinant protein per liter of growth medium. Specific selenium incorporation was observed only when constructs contained the wild type hTrxR1 gene, an E. coli SECIS element, and E. coli strains containing the native trans element of selenocysteine incorporation, selenophosphate synthetase (selD), were used for expression. Termination at the TGA codon was shown by mass spectrometry to be the cause of low incorporation of selenium. Elimination of the selenocysteine in the truncated enzyme inhibited overall catalytic activity and reduced the affinity for substrate. The Secys/Cys mutant exhibited about 5-10% of the activity of wild type rat liver TrxR1. N-terminal active site disulfide formation in the Secys/Cys mutant induced by oxidized TrxR1 substrate (thioredoxin) was about 12% that of wild type rat liver TrxR1 as measured by HPLC. These findings underscore the importance of the selenocysteine residue in thioredoxin reductase for catalysis. Cysteine cannot substitute effectively for selenocysteine. Selenium is a better nucleophile than sulfur and adjacent cysteine residues form strained disulfide bridges because of steric restrictions imposed by the large distance between the sulfur atoms, while selenocysteine-cysteine adjacent pairs are able to form a less strained selenosulfide linkage.